Although MIR516A has been reported to be downregulated and act as a tumor suppressor in multiple cancers, its expression and potential contribution to human bladder cancer (BC) remain unexplored. Unexpectedly, we showed here that MIR516A was markedly upregulated in human BC tissues and cell lines, while inhibition of MIR516A expression attenuated BC cell monolayer growth in vitro and xenograft tumor growth in vivo, accompanied with increased expression of PHLPP2. Further studies showed that MIR516A was able to directly bind to the 3'-untranslated region of PHLPP2 mRNA, which was essential for its attenuating PHLPP2 expression. The knockdown of PHLPP2 expression in MIR516A-inhibited cells could reverse BC cell growth, suggesting that PHLPP2 is a MIR516A downstream mediator responsible for MIR516A oncogenic effect. PHLPP2 was able to mediate BECN1/Beclin1 stabilization indirectly, therefore promoting BECN1-dependent macroautophagy/autophagy, and inhibiting BC tumor cell growth. In addition, our results indicated that the increased autophagy by attenuating MIR516A resulted in a dramatic inhibition of xenograft tumor formation in vivo. Collectively, our results reveal that MIR516A has a novel oncogenic function in BC growth by directing binding to PHLPP2 3'-UTR and inhibiting PHLPP2 expression, in turn at least partly promoting CUL4A-mediated BECN1 protein degradation, thereby attenuating autophagy and promoting BC growth, which is a distinct function of MIR516A identified in other cancers.Abbreviation: ATG3: autophagy related 3; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG12: autophagy related 12; BAF: bafilomycin A1; BC: bladder cancer; CHX: cycloheximide; Co-IP: co-immunoprecipitation; CUL3: cullin 3; CUL4A: cullin 4A; CUL4B: cullin 4B; IF: immunofluorescence: IHC-p: immunohistochemistry-paraffin; MIR516A: microRNA 516a (microRNA 516a1 and microRNA 516a2); MS: mass spectrometry; PHLPP2: PH domain and leucine rich repeat protein phosphatase.

中文翻译：

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MIR516A 在人膀胱癌中的致癌作用是由其减弱 PHLPP2 表达和 BECN1 依赖性自噬介导的。

尽管据报道 MIR516A 被下调并在多种癌症中充当肿瘤抑制因子，但其表达和对人类膀胱癌 (BC) 的潜在贡献仍未得到探索。出乎意料的是，我们在这里发现 MIR516A 在人 BC 组织和细胞系中显着上调，而 MIR516A 表达的抑制减弱了体外 BC 细胞单层生长和体内异种移植肿瘤的生长，伴随着 PHLPP2 的表达增加。进一步的研究表明，MIR516A 能够直接结合 PHLPP2 mRNA 的 3'-非翻译区，这对其减弱 PHLPP2 表达至关重要。MIR516A 抑制细胞中 PHLPP2 表达的敲低可以逆转 BC 细胞的生长，表明 PHLPP2 是 MIR516A 下游介质，负责 MIR516A 致癌作用。PHLPP2 能够间接介导 BECN1/Beclin1 的稳定，从而促进 BECN1 依赖性巨自噬/自噬，并抑制 BC 肿瘤细胞的生长。此外，我们的结果表明，通过减弱 MIR516A 增加的自噬导致体内异种移植肿瘤形成的显着抑制。总的来说，我们的结果表明，MIR516A 通过指导与 PHLPP2 3'-UTR 结合并抑制 PHLPP2 表达，进而至少部分促进 CUL4A 介导的 BECN1 蛋白降解，从而减弱自噬并促进 BC 生长，从而在 BC 生长中具有新的致癌功能，这是在其他癌症中鉴定的 MIR516A 的独特功能。缩写：ATG3：自噬相关 3；ATG5：自噬相关5；ATG7：自噬相关7；ATG12：自噬相关12；BAF：巴弗洛霉素A1；BC：膀胱癌；CHX：放线菌酮；Co-IP：共免疫沉淀；CUL3：cullin 3；CUL4A：库林4A；CUL4B：库林4B；IF：免疫荧光：IHC-p：免疫组化-石蜡；MIR516A：微小RNA 516a（微小RNA 516a1和微小RNA 516a2）；MS：质谱；PHLPP2：PH 结构域和富含亮氨酸的重复蛋白磷酸酶。